Trans-excision-splicing ribozyme and methods of use

ABSTRACT

A group I intron-derived ribozyme which binds RNA in trans, excises an internal segment from within the RNA, and splices the remaining 5′ and 3′ ends of the RNA back together (the trans-excision-splicing reaction) is disclosed. The excised segment can be as long as 28 nucleotides, or more, and as little as one nucleotide. The ribozymes of the invention are easily modified to alter their sequence specificity. Such ribozymes represent a new and potentially powerful class of generally adaptable genetic therapeutics.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/730,261, filed onDec. 9, 2003 now U.S. Pat. No. 7,211,661, which in turn claims thebenefit of U.S. Provisional Application No. 60/431,965, filed on Dec.10, 2002, the disclosures of which Applications are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates generally to the field of genetic therapeutics.More particularly, the invention relates to a trans-excision-splicingribozyme having adaptable sequence recognition specificity that providesa powerful tool for genetic therapies.

BACKGROUND OF THE INVENTION

The discovery of catalytic RNA fundamentally changed the course ofscience. The subsequent realization that catalytic RNAs could betailored to suit individual needs has been nothing less than inspiring.Indeed, the past ten years has seen the creative development of numerousRNA catalysts. Concurrently, the diversity of applications for thesecatalytic RNAs has been escalating. For example, catalytic RNAs arebeing developed for detection protocols, for therapeutic intervention ofdiseases, and for use as biochemical tools. As we continue to exploitthe steadily increasing knowledge base of RNA structure, folding, andcatalysis, designing and applying novel and effective RNA catalysts isbecoming more and more tractable.

Ribozymes are RNA molecules having an enzymatic activity, which enablesthe ribozyme to repeatedly cleave other separate RNA molecules in anucleotide base sequence-specific manner. Such enzymatic RNA moleculescan be targeted to virtually any RNA transcript, and efficient cleavageachieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987;Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988;and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme, which is held inclose proximity to an enzymatic portion of the RNA that acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA destroys its ability to direct synthesisof an encoded protein. After a ribozyme has bound and cleaved its RNAtarget it is released from that RNA to search for another target and canrepeatedly bind and cleave new targets.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofan enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

A catalytic RNA that can excise a specific RNA sequence out of a largerRNA (the trans-excision-splicing reaction), although not previouslydiscovered or engineered, would be very useful as a biochemical tool andalso as a potential new therapeutic strategy. For example, multipleturnover catalytic RNAs, or ribozymes, with this activity could be usedto excise a disease-causing RNA region out of a native transcript, toremove a premature stop codon, or to restore a frameshift mutation, forexample.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided atrans-excision-splicing ribozyme comprising at least two recognitionelements that are complementary to a target sequence within a substrate,wherein the target sequence is not complementary to native recognitionelements, wherein at least one of said recognition elements stabilizesbinding of the ribozyme to a trans-excision splicing (TES) reactionintermediate product, and wherein the ribozyme catalyzes a specificexcision of the target sequence and splices the 5′ end of the substratecreated by the excision to an ωG of the 3′ end of the substrate createdby the excision.

In another aspect of the invention there is provided a polynucleotidemolecule comprising a ribozyme expression cassette that is capable ofbeing stably inserted into a host, the cassette comprising a promoteroperably-linked to a nucleotide sequence encoding atrans-excision-splicing ribozyme comprising at least two recognitionelements that are complementary to target sequence within a substrateother than that complementary to native recognition elements, wherein atleast one of said recognition elements stabilizes binding of theribozyme to a trans-excision splicing (TES) reaction intermediateproduct and wherein the ribozyme catalyzes a specific excision of thetarget sequence and splices the 5′ end of the substrate created by theexcision to an ωG of the 3′ end of the substrate created by theexcision.

Also provided are host cells transfected with the polynucleotidemolecule.

In yet another aspect of the invention there is provided a method for invitro trans-splicing-excison of a target sequence, comprising the stepsof:

(1) providing the ribozyme of the invention in a trans-splicing reactionmixture;

(2) providing a substrate comprising the target sequence to the reactionmixture; and

(3) catalyzing the trans-splicing-excision of the target sequence.

In another aspect of the invention there is provided a method fordeleting an undesired genetic sequence from a host cell in vivo, saidmethod comprising:

(1) providing the ribozyme of the invention to the host cell, saidribozyme possessing catalytic activity against a target RNA sequencepresent in said host cell,

wherein the ribozyme catalyzes a specific excision of the target RNAsequence and splices the 5′ end of the substrate created by the excisionto an ωG of the 3′ end of the substrate created by the excision.

The following definitions are used herein.

Ribozyme: An RNA molecule that inherently possesses catalytic activity.

Trans-splice: A form of genetic manipulation whereby a nucleic acidsequence of a first polynucleotide is colinearly linked to or insertedcolinearly into the sequence of a second polynucleotide, in a mannerthat retains the 3′-5′ phosphodiester linkage between suchpolynucleotides. By “directed” trans-splicing or “substrate-specific”trans-splicing is meant a trans-splicing reaction that requires aspecific specie of RNA as a substrate for the trans-splicing reaction(that is, a specific specie of RNA in which to splice the transposedsequence). Directed trans-splicing may target more than one RNA specieif the ribozyme is designed to be directed against a target sequencepresent in a related set of RNAs.Target sequence: A nucleic acid molecule, e.g., RNA, that is a substratefor the catalytic activity of a ribozyme of the invention.Expression Cassette: A genetic sequence that provides sequencesnecessary for the expression of a ribozyme of the invention.Stably: By “stably” inserting a sequence into a genome is intendedinsertion in a manner that results in inheritance of such sequence incopies of such genome.Operable linkage: An “operable linkage” is a linkage in which a sequenceis connected to another sequence (or sequences) in such a way as to becapable of altering the functioning of the sequence (or sequences). Forexample, by operably linking a ribozyme encoding sequence to a promoter,expression of the ribozyme encoding sequence is placed under theinfluence or control of that promoter. Two nucleic acid sequences, suchas a ribozyme encoding sequence and a promoter region sequence at the 5′end of the encoding sequence, are said to be operably linked ifinduction of promoter function results in the transcription of theribozyme encoding sequence and if the nature of the linkage between thetwo sequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the expression regulatorysequences to direct the expression of the ribozyme. Thus, a promoterregion would be operably linked to a nucleic acid sequence if thepromoter were capable of effecting the synthesis of that nucleic acidsequence.Native target sequence/Non-native target sequence: Native targetsequence of a ribozyme is that polynucleotide sequence which isrecognized, bound and reacted by wild-type (native) ribozymes.Non-native target sequence is sequence within a substrate that is notbound by wild type ribozyme and consequently native ribozyme does notreact with non-native target sequence. Non-native target sequence canoccur as a result of mutation, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The Group I Intron Self-Splicing (1 a: left) andTrans-Excision-Splicing Reactions (1 b: right). The catalytic RNAs arerepresented by gray lines, the 5′ and 3′ exons and mimics are blacklines, and the bridge (excised region) is a dotted line. The circle inthe 5′ exon represents a uridine and the circle in the 3′ exonrepresents a guanosine. The intron inherently contains all the requiredactivities for the trans-excision-splicing reaction. RE1, RE2, and RE3are three recognition elements that the catalytic RNAs may use to basepair with their substrates (RE3 is also referred to as the IGS, orInternal Guide Sequence.). It is likely that RE2 and/or RE3 do not bindthe substrate until after the first reaction step. In addition, at leastwith the rP-8/4x ribozyme used herein, the guanosine cofactor is notrequired for initiation of the first catalytic step.

FIG. 2. The rP-8/4x and rP-8/4x-MD Ribozymes Base Pairing with VariousSubstrates. 2A) The P. carinii ribozyme, rP-8/4x (uppercase lettering,SEQ ID NO:9), binding to the 36-mer substrate (lowercase lettering witha gray background, SEQ ID NO:10). In the trans-excision-splicingreaction, the bridging region (white lettering) is excised and the 5′and 3′ regions of the substrate (black lettering) are subsequentlyspliced together. Note that P1, P9.0, and P10 are helices that resultfrom the recognition elements RE1, RE2, and RE3 base pairing with thesubstrate. The large bold arrows indicate the sites of catalysis for thefirst (left) and second (right) step of the trans-excision-splicingreaction. The 5′ uridine and 3′ guanosine are circled. The ribozymebases are numbered according to that for the P. carinii intron (Testa,S. M., Haidaris, C. G., Gigliotti, F., & Turner, D. H. (1997)Biochemistry 36, 15303-15314.13, incorporated herein in its entirety).2B). Simplified diagrams of various substrates base pairing with variousribozymes. Only the recognition element sequences are shown for theribozymes. The dashed line indicates a normal phosphodiester bondbetween the adjoining sequences. i) The 12-mer substrate (SEQ ID NO:7)binding to rP-8/4x (SEQ ID NO:11). ii) The 10-mer substrate (SEQ IDNO:8) binding to rP-8/4x (SEQ ID NO:11). iii) The 38-mer MyotonicDystrophy substrate (SEQ ID NO:12) binding to the rP8/4x-MD MyotonicDystrophy ribozyme (SEQ ID NO:13).

FIG. 3. The Trans-Excision-Splicing Reaction using the P. cariniisystem. The Trans-Excision-Splicing (TES) Reaction Using the P. cariniiSystem. A) Polyacrylamide gel showing substrates and products of the TESreaction using 166 nM rP-8/4x ribozyme and 1.33 nM substrate at 7 mMMgCl₂ (36-mer) and 10 mM MgCl₂ (10-mer and 12-mer) at 44° C. Thereaction using the 36-mer substrate is diagrammed on the left (SEQ IDNO:10, SEQ ID NO:14, and SEQ ID NO:15). The regions of the substratethat bind to the ribozyme's recognition elements (labeled RE1 (SEQ IDNO:15), RE2, and RE3) are underlined. All reactions in the presence (+)and absence (−) of the rP-8/4x ribozyme were subjected to the sameincubation conditions. TES reactions were conducted using a 36-mersubstrate (to give a 16-mer product), a 12-mer substrate (9-merproduct), and a 10-mer substrate (9-mer product). See FIG. 2 for thesequence of these substrates. The 6-mer lane shows a synthetic controlfor the 5′ cleavage products, the 16-mer lane shows a synthetic controlfor the 16-mer TES product, and the —OH lane shows an alkaline digest ofthe 36-mer starting material. B). Graphs of TES reactions using the36-mer substrate. All reactions were run as above except for thechanging variable. The TES product is represented by filled circles andthe 5′ cleavage product by open circles. C). Graphs of TES reactionsusing the 10-mer substrate. All reactions were run as above except forthe changing variable. The TES product is represented by filledtriangles and the 5′ cleavage product by open triangles. Each graphshows the average of two independent assays.

FIG. 4. Sequencing of Trans-Excision-Splicing Products. For each system,a chemically synthesized version of the expected product was sequencedand run adjacent to the isolated excision product. Left: the P. carinii16-mer product that results from treating the 36-mer substrate with therP-8/4x ribozyme. Right: the Myotonic Dystrophy 10-mer product thatresults from treating the 38-mer substrate with the rP-8/4x-MD ribozyme.The dotted line represents the newly created splice junction between the5′ and 3′ ends of the substrate. Nuclease T1 is specific for guanosine,U2 for adenosine, and CL-3 primarily for cytidine. The ⁻OH lane shows analkaline digest of the starting material, and the lanes labeled ‘Start’show the starting material.

FIG. 5. Competition TES Reactions. Polyacrylamide gel showing substratesand products of TES reactions using 166 nM rP-8/4x, 7 mM MgCl₂, and 44°C. ‘Radiolabeled substrate’ refers to the length of the radiolabeledsubstrate at 1.33 nM final concentration and ‘cold substrate’ refers tothe length of the non-radiolabeled substrate. The final concentrationsof the cold substrates are 1.33 nM for the 36-mer, and 66.5 nM (50×) or665 nM (500×) for the 7-mer, which is r(GUGCUCU) (SEQ ID NO:16). The twosubstrates for each reaction were added simultaneously. Lanes designatedin the first box are length controls, the second box shows the 5′ exoncompetition assay, the third box shows an alkaline digest of the 36-merstarting material, and the fourth box shows the 3′ exon competitionassay.

FIG. 6. The TES Reaction Using the DMPK Model System. A) Polyacrylamidegel showing substrates and products of the TES reaction using 166 nMrP-8/4x-MD ribozyme, 1.33 nM substrate, 13 mM MgCl₂, and 44° C. Thereaction using the 38-mer DMPK mimic is diagrammed on the left (SEQ IDNO:12, SEQ ID NO:17 and SEQ ID NO:18). The regions of the substrate thatbind to the ribozyme's recognition elements are labeled RE1 (SEQ IDNO:18), RE2, and RE3. All reactions in the presence (+) and absence (−)of the rP-8/4x-MD ribozyme were subjected to the same incubationconditions. TES reactions were conducted using the 38-mer substrate (togive a 10-mer product). The 6-mer lane shows a synthetic control for the5′ cleavage products, the 10-mer lane shows a synthetic control for the10-mer TES product, and the —OH lane shows an alkaline digest of the38-mer starting material. The lanes labeled 36 are TES reactions usingthe rP-8/4x-MD ribozyme with the 36-mer P. carinii substrate (at 13 mMMgCl₂), and the lane labeled 38 (lane o) is a reaction using the rP-8/4xribozyme with the 38-mer DMPK mimic (at 7 mM MgCl₂). In these cases, noreaction occurs. B). Graphs of TES reactions using the 38-mer substrateand rP-8/4x-MD. All reactions were run as above except for the changingvariable. The TES product is represented by filled squares and the 5′cleavage product by open squares.

FIG. 7. TES reactions using two different Tetrahymena ribozymes. FIG. 7Ais a polyacrylamide gel showing substrates, intermediates, and productsof the TES reaction. The reaction was carried out for 90 minutes using166 nM ribozyme and 1.33 nM radiolabeled 13-mer substrate at either 0 mMor 10 mM MgCl₂ at 44 degrees C., either in the presence (+) or absence(−) of 330 nM GMP. FIG. B is a diagram of the TES reaction (SEQ ID NO:19and SEQ ID NO:20). The excised G of SEQ ID NO:19 is in bold lettering.FIG. 7C shows two graphs of TES reactions using the 13-mer substrate andthe A-L-21 Sca ribozyme (Tetrahymena). The TES product is represented bythe filled circles and the 5′ intermediate (CUCUCU) (SEQ ID NO:21) isrepresented by open circles. All reactions were run as above except forthe changing variable. Each curve represents the average of twoindependent assays. Standard deviation for all points was less than 10%.For clarity the graphs use different scales.

DETAILED DESCRIPTION OF THE INVENTION

The inherent binding and catalytic activity of group I intron-derivedribozymes can be exploited to catalyze the trans-excision-splicingreaction. FIG. 1 a shows a simple diagram of a typical group Iintron-catalyzed self-splicing reaction. There are three base pairingcontacts that the intron uses to hold and position its 5′ and 3′ exonsfor subsequent catalysis. We have termed these the ribozyme recognitionelements, or RE1, RE2, and RE3. Physically removing the intramolecularexons from the intron creates a ribozyme that has the ability to bindand catalyze reactions using exogenous substrates that contain 5′ and 3′exon sequences (Zaug, A. J. & Cech, T. R. (1986) Science 231, 470-475.).If the 5′ and 3′ exon sequences are connected with a bridging sequenceand recognition elements are maintained, this ribozyme can excise thebridging sequence and splice the 5′ and 3′ ends of the substratetogether. A simple diagram of this trans-excision-splicing reaction isshown in FIG. 1 b. This reaction was tested using a ribozyme, rP-8/4x,from the opportunistic pathogen Pneumocystis carinii. In FIG. 2A it isshown that this ribozyme catalyzes the trans-excision-splicing reactionusing a synthetic substrate which is complementary to the nativerecognition elements of the rP-8/4x ribozyme. Moreover, as little as asingle nucleotide can be excised.

In order for this reaction to be useful, however, the ribozymerecognition elements have to be mutable, such that the ribozyme can betargeted to bind with and react on non-native sequences. Using a group Iintron-derived ribozyme from Tetrahymena thermophila, the sequence ofRE1 (also referred to as the IGS, or Internal Guide Sequence) wasmutated and the resultant ribozyme then bound to a new, complementarysubstrate (Murphy, F. L. & Cech, T. R. (1989) Proc. Natl. Acad. Sci.U.S.A. 86, 9218-9222; Been, M. D. & Cech, T. R. (1986) Cell 47, 207-216;Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature 324, 429-433).This property has been exploited for, among other purposes, designingtrans-splicing ribozymes that can replace the 3′ end of mutanttranscripts with corrected versions (Sullenger, B. A. & Cech, T. R.(1994) Nature 371, 619-6; Lan, N., Howrey, R. P., Lee, S. W., Smith, C.A., & Sullenger, B. A. (1998) Science 280, 1593-1596).

Trans-excision-splicing ribozymes are ideal for treating geneticdiseases for which the causative affects may be ameliorated by theexcision of an internal RNA segment out of a larger RNA, includingtriplet-expansion diseases such as Huntington's disease, Fragile X, andMyotonic Dystrophy (Bowater, R. P. & Wells, R. D. (2001) Prog. NucleicAcid Res. Mol. Biol. 66, 159-202). Patients affected with these diseaseshave an RNA that is present in normal individuals, except that embeddedwithin it is an abnormally high number of tandem repeat sequences. ForMyotonic Dystrophy, the expansion is in the 3′ UnTranslated Region [3′UTR] of a serine-threonine protein kinase gene [the DMPK gene], whoseexpression induces the expression of skeletal muscle specific genes. Thedisease state of Myotonic Dystrophy, which is the most common form ofadult onset Muscular Dystrophy, typically has much more than 35 CUGrepeats, while unaffected individuals typically have less than 15repeats. The greater the number of repeats, the more severe the affectsof the disease. Moreover, strong experimental evidence indicates thedisease stems directly from the RNA repeats, and not to any affectedcoding potential of the parent DNA. In fact, the Myotonic Dystrophytriplet-expansion and recently discovered quadruplet-expansion diseaseshave the repeat sequences within non-coding regions, so their effectsare not due to the synthesis of mutant proteins. A new class ofribozymes that can specifically excise these expanded RNA repeats fromthe transcripts could aid in the development of much needed therapeuticsagainst these types of diseases.

The rP-8/4x ribozyme was re-engineered to test the potential to alterthe sequence specificity of trans-excision-splicing ribozymes to bindand excise the triplet expansion region from a Myotonic Dystrophy DMPKsmall model system in vitro. The resultant ribozyme excises the tripletexpansion region, and in a sequence specific manner. Thattrans-excision-splicing ribozymes can be re-engineered to target and actupon predetermined sequences demonstrates a general usefulness for theseribozymes as biochemical tools and therapeutics.

P. carinii Group I Intron Ribozyme Catalyzes the Trans-Excision-SplicingReaction.

A ribozyme derived from a P. carinii group I intron catalyzes apreviously unreported excision-splicing reaction on an exogenoussubstrate. This trans-excision-splicing reaction takes advantage of thecatalytic abilities of this ribozyme. First, rapid and efficientsequence-specific cleavage of a designated substrate occurs in aguanosine independent fashion, as previously reported (Testa, S. M.,Haidaris, C. G., Gigliotti, F., & Turner, D. H. (1997) Biochemistry 36,15303-15314.). Second, the resultant 5′ cleavage product can splice to asequence that binds the 3′ end of the ribozyme (Inoue, T., Sullivan, F.X., & Cech, T. R. (1985) Cell 43, 431-437) (FIG. 1B). These activitieswere exploited by designing a synthetic substrate that contains withinit the sequences required for each catalytic event, separated by aninternal bridging sequence (FIG. 2A). As seen in FIG. 3, the engineeredribozyme catalyzes the specific excision of the bridge sequence andsplices the 5′ and 3′ ends of the substrate back together. Even thoughthere are 18 other uridines that could be sites of 5′ cleavage and 4other guanines that could be sites of 3′ splicing, only onetrans-excision-splicing product is generated. Apparently, the ribozymerecognition elements that define the individual catalytic steps aresequence specific.

That the ribozyme can catalyze this reaction is surprising in that therelatively long bridging region could be expected to sterically hinderthe binding of the substrate to the catalytic core of the ribozyme orhinder the required conformational rearrangement between the twocatalytic steps (Cech, T. R., Herschlag, D., Piccirilli, J. A., & Pyle,A. M. (1992) J. Biol. Chem. 267, 17479-17482; and 39. Jaeger, L.,Michel, F. & Westhof, E. (1996) in Catalytic RNA, eds. Eckstein, F. &Lilley, D. (Heidelberg, Germany), Vol. 10, pp. 1-17). Perhaps thisaccounts for over 50% of the substrate only undergoing the firstcatalytic step (see FIG. 3). In addition, like other ribozymes, the P.carinii ribozyme binds its 5′ exon sequences orders of magnitude tighterthan its 3′ exon sequences, which allows time for 3′ end dissociationprior to the second catalytic step. Nevertheless, a significant amountof trans-excision-splicing product is generated. Another considerationis that the 5′ exon of one substrate could ligate to the 3′ exon ofanother substrate. Since spontaneous 3′ splice site hydrolysis isessentially non-existent and only an insignificant amount of 5′exon-bridge 26-mer product (which would be a side reaction of thiscatalytic event) is produced, it is unlikely that this mechanism occursto a significant extent.

The P. carinii ribozyme can excise as little as one nucleotide. The P.carinii ribozyme excises as little as a single nucleotide, and in asequence-specific manner. Apparently, there is no lower limit to thesize of the region being excised. That the same approximate yield isobtained regardless of whether the 12-mer (39%) and 10-mer (36%)substrate is used suggests that the role of forming the P9.0 helix isnot large in this case, as the 10-mer lacks the ability to form the P9.0helix. Therefore, the RE2 interaction, although perhaps beneficial, isnot required to establish a sequence specific interaction. In addition,the 12-mer and 10-mer substrates lead to approximately 50% more productas compared with the 36-mer substrate. There are many possibleexplanations for this, including the longer bridge of the 36-mersubstrate partially interfering with the substrate's ability to bind therecognition elements.

The sequence specificity of the P. carinii ribozyme can be altered.Altering the recognition elements of the P. carinii ribozyme changes thesequence specificity of the trans-excision-splicing reaction. While itwas known that RE1 could be modified in reactions mimicking the firststep of the self-splicing reaction, it was previously not known that allthree elements are mutable. That these recognition elements completelyspecify binding and reactivity indicates that they are the primarydeterminants of specificity between the ribozyme and its substrate. Inaddition, bridging regions of different sequences and lengths (1, 3, 20,and 28 nucleotides) have been excised, indicating that the 3′ G in thebridging region might be the only potential sequence requirement for theexcised segment. Structure might even be tolerated within the excisedregion, as the somewhat structured CUG repeat within the MyotonicDystrophy substrate doesn't prohibit the reaction. In addition, eachribozyme only acts upon its designated target, even though the RE1s inboth ribozymes are 50% identical, giving a further indication of thehigh level of sequence specificity of this reaction. Although the MgCl₂concentration required for maximum activity is different for the tworibozymes (7 and 13 mM), they both are active throughout the samegeneral MgCl₂ concentration range (data not shown), which is below thatrequired (15 mM) for maximum activity for the P. carinii group I intronself-splicing reaction in vitro.

The ability to catalyze TES reactions is inherent to Group I introns. Todetermine whether other ribozymes can catalyze TES reaction, aTetrahymena-containing L-21 ScaI plasmid was linearized with ScaI,run-off transcriptions were performed, and the ribozyme was purified asdescribed by Testa et al. (Biochemistry, (1997) 36:15303-15314). TESreactions were run in a variety of standard buffers and conditionsroutinely used for ribozyme reactions. The reactions were also run inthe presence and absence of the PG cofactor. In all cases, the firststep of the reaction was the only reaction step that was detected.

The lack of a full-length, second-step reaction product suggests thatthe second step may be inefficient or problematic with this ribozyme.Our previous studies of the P. carinii ribozyme had demonstrated that 3′intermediate disassociation results in significantly reduced second-stepyields. Therefore, the Tetrahymena ribozyme was modified to contain anRE3 region in order to stabilize binding of the ribozyme to the '3 exonreaction intermediate. The results are shown in FIG. 6. The reactionusing the stabilized ribozyme generates a product band of the expectedsize (a 12-mer product from a 13-mer substrate). The product band wasexcised from the band and sequenced, and shown to be the expected TESproduct. These results demonstrate that the ability to catalyze TESreactions is a general property inherent to group I introns.

Comparison with the Trans-Splicing Reaction. The trans-excision-splicingreaction of the invention is fundamentally different from thetrans-splicing reaction previously reported (Sullenger, B. A. & Cech, T.R. (1994) Nature 371, 619-622.), whereby a ribozyme covalently attachedto a normal RNA sequence binds to a mutant RNA transcript, cleaves themutant transcript, and replaces the 3′ end of the mutant transcript withthe normal ‘corrected’ version. While trans-splicing can correct mutantRNA, it is single-turnover, it exploits only the RE1 molecularinteraction, and the ribozyme must be covalently attached to the new,corrected transcript. In contrast, the trans-excision-splicing reactionis potentially multiple turnover; it exploits the RE1, RE2, and/or RE3molecular interactions; and it excises an internal segment from within alarger RNA. Nevertheless, these two complementary reactions share manysimilarities and so the wealth of knowledge already reported for thetrans-splicing reaction is applicable to the trans-excision-splicingreaction.

Implications for Myotonic Dystrophy. The rP-8/4x ribozyme can beredesigned to specifically excise the entire triplet expansion region ofa mimic of the Myotonic Dystrophy transcript, implicating such ribozymesas new types of potential Muscular Dystrophy therapeutics. As apractical matter, cutting out this entire region may or may not restoreproper function to the transcript, as normal individuals typically haveat least 5 repeats. Similar multiple turnover ribozymes, however, can bedesigned to target the repeats themselves to successively reduceexpansion lengths.

General Therapeutic Implications. Unfortunately, nature has afforded ahuge number of RNA-mediated mutations that predispose individuals todisease. New therapeutic strategies to combat these diseases are needed.That the recognition elements of the rP-8/4x ribozyme can be modifiedand the size and sequence of the excised region do not appear to havelimitations suggest that trans-excision-splicing ribozymes may begenerally useful as therapeutics against many such diseases. Forexample, excising specific sequences could remove premature stop codons,restore altered reading frames, or remove insertion mutations thataffect transcription and translation regulation.

Trans-excision-splicing ribozymes are thought to have two sequencepreferences. The first is a uridine at the 5′ cleavage site (although acytidine might also work) and the second is a guanosine at the 3′ splicesite (FIG. 2A). Therefore, there are three simple targeting strategiesto consider. First, a ribozyme can be designed that targets a uridine 5′to any particular mutation and a guanosine 3′ to this mutation, as isthe case in the Myotonic Dystrophy model system described here. Itshould be noted that if more than an insertion mutation is excised thenew transcript might not function normally. Second, a mutation thatresults in a new guanosine can be targeted as the 3′ splice site. Theabove strategies will also target normal transcripts for at least thefirst step of the reaction, so these strategies should be designed withcare. Third, a mutation that results in a new uridine can be targeted asthe 5′ cleavage site. This resultant ribozyme would not attack thenormal transcript, but would perform both steps on the mutanttranscript. Since point mutations that result in premature terminationcodons often involve a new uridine (all termination codons begin withuridine), hundreds of distinct mutations could be specifically targeted.Therefore, many options for targeting are possible, which expands thepotential usefulness of these ribozymes. The general strategy oftargeting mutations at the RNA level, however, should be considered apotential treatment rather than a potential cure, as mutant RNAtranscripts will continue to be produced.

As shown herein, group I intron-derived ribozymes obtained fromdifferent organisms can catalyze this new trans-excision-splicingreaction. In addition, the sequence of the ribozyme can be easilymanipulated such that it targets and acts upon desired substrates,including those of medical importance. The reactions themselves appearhighly sequence specific and as little as a single nucleotide can beexcised. The applicability of trans-excision-splicing ribozymes to treatdisease has been demonstrated by designing a ribozyme that specificallyremoves the triplet expansion region that is involved in a common formof Muscular Dystrophy in a small model system in vitro. Therefore,trans-excision-splicing ribozymes are a new class of ribozymes thatpermits potential biochemical and therapeutic strategies not beforepossible.

The ribozymes of the invention can be introduced into and expressed in ahost cell. Transcription of the ribozyme of the invention in a host celloccurs after introduction of the ribozyme gene into the host cell. Ifthe stable retention of the ribozyme by the host cell is not desired,the ribozyme may be chemically or enzymatically synthesized and providedto the host cell by mechanical methods, such as microinjection,liposome-mediated transfection, electroporation, calcium phosphateprecipitation, or the like. Alternatively, when stable retention of thegene encoding the ribozyme is desired, such retention may be achieved bystably inserting at least one DNA copy of the ribozyme into the host'schromosome, or by providing a DNA copy of the ribozyme on a plasmid thatis stably retained by the host cell. Preferably the ribozyme of theinvention is inserted into the host's chromosome as part of anexpression cassette, which provides transcriptional regulatory elementsthat control the transcription of the ribozyme in the host cell. Suchelements may include, but not necessarily be limited to, a promoterelement, an enhancer or UAS element, and a transcriptional terminatorsignal. Polyadenylation is not necessary as the ribozyme is nottranslated.

Expression of a ribozyme whose coding sequence has been stably insertedinto a host's chromosome is controlled by the promoter sequence that isoperably linked to the ribozyme coding sequences. The promoter thatdirects expression of the ribozyme may be any promoter functional in thehost cell, prokaryotic promoters being desired in prokaryotic cells andeukaryotic promoters in eukaryotic cells. A promoter is composed ofdiscrete modules that direct the transcriptional activation and/orrepression of the promoter in the host cell. Such modules may be mixedand matched in the ribozyme's promoter so as to provide for the properexpression of the ribozyme in the host. A eukaryotic promoter may be anypromoter functional in eukaryotic cells, and especially may be any of anRNA polymerase I, II or III specifically. If it is desired to expressthe ribozyme in a wide variety of eukaryotic host cells, a promoterfunctional in most eukaryotic host cells should be selected, such as arRNA or a tRNA promoter, or the promoter for a widely expressed mRNAsuch as the promoter for an actin gene, or a glycolytic gene. If it isdesired to express the ribozyme only in a certain cell or tissue type, acell-specific (or tissue-specific) promoter element that is functionalonly in that cell or tissue type should be selected.

The trans-splicing reaction is chemically the same whether it isperformed in vitro or in vivo. However, in vivo, the presence of thetarget and the ribozyme will suffice to result in trans-splicing, sincecofactors are usually already present in the host cell.

It has been previously reported that, at high MgCl₂ (10-100 mM) andtemperature (55° C. to 65° C.), Tetrahymena group I intron ribozymesthat lack both of the exons and the IGS can catalyze a guanosinecofactor-mediated TES-like reaction upon binding pseudoknot structuredsubstrates, which creates the in trans equivalent of the P1 and P10helices (P9.0 helix formation was not required). The ribozymes targetand bind these pseudoknot structures entirely through tertiaryinteractions. In contrast, the ribozymes used in the present inventioncontain at least two modifiable REs. In a preferred embodiment, theribozyme contains modified RE1 and RE3. which allows the ribozyme totarget designated substrates at the level of simple base pairing.Furthermore, the TES reactions using the P. carinii and Tetrahymenaribozymes do not require a guanosine cofactor, and optimally occur at alower MgCl₂ concentration (7-13 mM) and temperature (44° C.).

Sullenger and Cech ((1994) Nature 371, 619-622.) previously reportedthat Tetrahymena group I intron ribozymes that lack a 5′ exon, butcontain an endogenous non-native 3′ exon, catalyze the covalentattachment of the endogenous 3′ exon to mutant transcripts in such a wayas to replace the 3′ end of mutant transcripts with normal ‘corrected’versions. While trans-splicing can repair RNA, it exploits only the RE1molecular interaction, the ribozymes must be covalently attached to therepaired half of the transcript, it is single-turnover, and repairingmutations distant from the 3′ end of long transcripts could beproblematic. In contrast, the TES reaction exploits multiple molecularinteractions (RE1, RE3, and perhaps RE2), TES ribozymes excise aninternal segment from within RNA substrates, the reaction is potentiallymultiple turnover, and the position of the mutations within thetranscript is not a limiting factor. Moreover, that under the conditionsused in this report, little (if any) turnover was observed.

Example 1

Oligonucleotide synthesis and preparation. DNA oligonucleotide primerswere purchased from Integrated DNA Technologies (Coralville, Iowa), andwere used without further purification. RNA oligonucleotides werepurchased from Dharmacon Research Inc. (Boulder, Colo.) and deprotectedfollowing the manufacturer's protocol. The oligoribonucleotides were 5′end radiolabeled and purified via gel electrophoresis as previouslydescribed (Testa, S. M., Haidaris, C. G., Gigliotti, F., & Turner, D. H.(1997) Biochemistry 36, 15303-15314.). The RNA products were extractedfrom the gel slice by stirring for one hour with a sterile stir-bar in1.5 mL elution buffer containing 10 mM Tris (pH 7.4), 250 mM NaCl, and 1mM EDTA. Gel particulate was removed via centrifugation, and thesolution was evaporated to a final oligoribonucleotide concentration ofapproximately 8 nM.

Plasmid construction and synthesis. The P. carinii ribozyme plasmidprecursor, P-8/4x, was generated as previously described (Testa, S. M.,Haidaris, C. G., Gigliotti, F., & Turner, D. H. (1997) Biochemistry 36,15303-15314). The Myotonic Dystrophy-specific ribozyme plasmidprecursor, P-8/4x-MD, was derived from the P-8/4x plasmid bysite-directed mutagenesis. Briefly, three successive rounds ofmutagenesis were performed to modify each of the three recognitionelements using the following pairs of mutagenic primers (underlinedbases represent altered recognition elements as compared to P-8/4x):^(5′)CACGCCGCTTTCGGGAACCTCTATAGTGAGTCG^(3′) (SEQ ID NO:1) and^(5′)CGACTCACTATAGAGGTTCCCGAAAGCGGCGTG^(3′) (SEQ ID NO:2) for RE1formation, ^(5′)GGTATAGTCTTGCCTCTTTCGAAAG^(3′) (SEQ ID NO:3) and^(5′)CTTTCGAAAGAGGCAAGACTATACC^(3′) for RE2 (SEQ ID NO:4) formation, andthen ^(5′)CGACTCACTATAGGTGTTCCCGAAAGCGGC^(3′) (SEQ ID NO:5) and^(5′)GCCGCTTTCGGGAACACCTATAGTGAGTCG^(3′) (SEQ ID NO:6) for RE3formation. Each set of primers (15 pmol each primer) was used in anamplification reaction comprising 25 ng parental plasmid, 2.5 units PfuDNA polymerase (Stratagene; La Jolla, Calif.), and 0.5 μM dNTPs in abuffer comprising 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.8), 2mM MgSO₄, 0.1% Triton X-100, and 0.1 mg/mL BSA (final volume 50 uL).After an initial denaturation for 30 seconds at 95° C., the mixture wassubjected to 15 cycles of 95° C. for 30 seconds, 50° C. for two minutes,and 68° C. for six minutes. Parental plasmid was then digested with 20units Dpn I (Gibco BRL; Rockville, Md.) in 5.7 μL of the manufacturer'ssupplied buffer for 2 hours at 37° C. 3 μL of this mixture was then usedto transform E. coli DH5α competent cells (Gibco BRL). The vectors werepurified using a QIAprep Spin Miniprep Kit (QIAGEN; Valencia, Calif.).The resultant final plasmid, P-8/4x-MD, was sequenced for confirmation(ACGT, Inc; Northbrook, Ill.). The plasmids were linearized with Xba I,phenol/chloroform extracted, and ethanol precipitated in preparation forrun-off transcription.

Transcription. Both rP-8/4x and rP-8/4x-MD were transcribed from theirappropriate plasmid precursors essentially as previously described forrP-8/4x (Testa, S. M., Haidaris, C. G., Gigliotti, F., & Turner, D. H.(1997) Biochemistry 36, 15303-15314). A typical transcription reaction(40 μL) contained 1 μg linearized plasmid, 40 mM Tris-HCl (pH 7.4), 5 mMdithiothreitol, 5 mM spermidine, 5 mM MgCl₂, 1.5 mM each NTP, 1.25 mg/mLBSA, and 4 μL of T7 RNA polymerase (100 units/μL) and was incubated fortwo hours at 37° C. The resultant RNA was purified using QIAGEN-tip 100anion-exchange columns. First, each column was equilibrated with 4.0 mLof Buffer I (750 mM NaCl, 50 mM MOPS (pH 7.0), 15% ethanol, and 0.15%Triton X-100). Second, the transcription reactions were loaded onto thecolumn and the column was washed with 7.0 mL of Buffer I. Third, thetranscripts were eluted using 4.0 mL of Buffer II (1.0 M NaCl, 50 mMMOPS (pH 7.0), and 15% ethanol). Following an isopropanol and then anethanol precipitation, the samples were dissolved in water andquantified using a Beckman UV-VIS DU-650 spectrophotometer.

Trans-excision-splicing reactions. Reactions were conducted in H×Mgbuffer consisting of 50 mM Hepes (25 mM Na⁺), 135 mM KCl, and x mM MgCl₂(listed in the figures) at pH 7.5. The trans-excision-splicing reactionswere optimized for the rP-8/4x and rP-8/4x-MD ribozymes over a MgCl₂concentration range of 0 to 50 mM at 30° C., 37° C., and 44° C. Maximumproduct formation occurred at 44° C. for both ribozymes, 7 mM MgCl₂ forrP-8/4x, and 13 mM MgCl₂ for rP-8/4x-MD, although a significant amountof each product was obtained at 37° C. at both 7 and 13 mM MgCl₂ foreach ribozyme (data not shown). Prior to each reaction, 1.0 pmol ofribozyme in 5.0 μL of the appropriate buffer was preannealed at 60° C.for five minutes and then allowed to slow-cool to the appropriatetemperature. Reactions using the rP-8/4x ribozyme were initiated byadding 1.0 μL of 8 nM radiolabeled 36-mer, 12-mer, or 10-mer P.carinii-specific substrates or the 38-mer Myotonic Dystrophy substrate.Reactions using the rP-8/4x-MD ribozyme were initiated by adding 1.0 μLof 8 nM radiolabeled 38-mer Myotonic Dystrophy-specific substrate or the36-mer P. carinii substrate. The substrate sequences and how they basepair with the ribozymes are shown in FIG. 2. In each case the substrateswere preincubated in the appropriate buffer (listed in the figures).After one hour, the reactions were terminated by adding an equal volumeof stop buffer (10 M urea, 3 mM EDTA, and 0.1×TBE). The products andreactants were denatured for one minute at 90° C. and then separated ona 12% acrylamide/8 M urea gel. The gel was transferred to chromatographypaper (Whatman 3MM CHR) and dried under vacuum. The bands werevisualized and quantified on a Molecular Dynamics Storm 860Phosphorimager.

The observed rate constant, k_(obs), for the first (5′ cleavage) andsecond (exon ligation) step of each reaction was quantified (Testa, S.M., Gryaznov, S. M. & Turner, D. H. (1998) Biochemistry 37, 9379-9385;Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C.,Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K., et al(1992) Science 255, 1253-1255). The first step was obtained from a plotof the percent intermediate plus percent product formed versus time, andthe second step was obtained from a plot of percent product formedversus time. These observed rate constants reflect the rate of chemistryand any requisite conformation changes that occur.

Sequencing the trans-excision-splicing products. Products obtained fromthe trans-excision-splicing reactions were gel purified and sequenced bypartial nuclease digestion using T1 (Epicentre; Madison, Wis.), U2(Research Unlimited; Wellington, New Zealand), and Cl-3 (ResearchUnlimited) RNA nucleases. T1 reactions used 1.0×1 units T1 in 200 mMTris-HCl (pH 7.5), Cl-3 reactions used 0.33 units Cl-3 in 200 mMTris-HCl (pH 7.5), and U2 reactions used 0.33 units U2 in 200 mMTris-HCl (pH 3.5). Sequencing reactions utilized approximately 50 fmolof RNA and were incubated for 10 minutes at 55° C. Immediately afteradding an equal volume of stop buffer to each reaction, aliquots wereloaded on a 13.5% polyacrylamide/8 M urea gel. In parallel with theabove reactions, we enzymatically sequenced chemically synthesizedversions of the expected trans-excision-splicing products forcomparison.

To determine if the 3′ product of the first reaction step (5′ cleavage)(FIG. 1B) is dissociating and then rebinding the same (or different)ribozyme before the second reaction step (exon ligation), TES reactionswere conducted for one hour in 7 mM MgCl₂, 166 nM rP-8/4x ribozyme, 1.33nM radiolabeled 36-mer substrate, and either 66.5 nM (50×) or 665 nM(500×) 3′ exon mimic competitor r(GUGCUCU) (SEQ ID NO:16). The valuesreported are the average of six independent assays. Likewise, todetermine if the 5′ product of the first reaction step (5′ cleavage)(FIG. 1B) is dissociating and then rebinding the ribozyme before thesecond step (exon ligation), TES reactions were conducted for one hr in7 mM MgCl₂, 166 nM rP-8/4x ribozyme, 1.33 nM 36-mer non-radiolabeledsubstrate, and 1.33 nM radiolabeled 5′ exon mimic competitor r(AUGACU)(SEQ ID NO:15). In each case the competitors were added simultaneouslywith the substrates.

Example 2

The P. carinii group I intron ribozyme catalyzes thetrans-excision-splicing reaction In order to test whether a ribozymederived from a group I intron, and specifically one from P. carinii,catalyzes the trans-excision-splicing reaction, a substrate was designedthat would bind the rP-8/4x ribozyme's native recognition elementsequences (RE1, RE2, and RE3 in FIG. 1 b). These substrate sequenceswere connected with a bridge consisting of the first four bases of theintron and 13 uridines (FIG. 2A). Uridines where chosen because of theirrelatively poor ability to form self-structures and the number ofuridines (13) was chosen arbitrarily. Typical results at the optimizedMgCl₂ concentration (7 mM) and the temperature (44° C.) are shown inFIG. 3A.

The expected product band at 16 nucleotides in length was obtained in ayield of 25%±5% (for 6 independently run assays). This band wasextracted from the gel matrix, repurified, and subjected to enzymaticsequencing, along with a chemically synthesized version of the expectedproduct. The sequence and banding patterns were identical (FIG. 4),indicating that the expected trans-excision-splicing product wasgenerating. The reaction also produced a band at six nucleotides. Thisis a product of the first step of the two-step trans-excision-splicingreaction (5′ cleavage), and indicates that a portion of the transcriptundergoes only the first step of the reaction. Also produced is a smallamount of ribozyme mediated 3′ splice site hydrolysis product at 26nucleotides. Nonetheless, the rP-8/4x ribozyme contains the ability tobind a substrate in trans and catalyze the excision-splicing reaction.

Apparently, a majority of the 36-mer substrate undergoes only the firststep of the reaction. The dependence of the TES reaction on MgCl₂concentration, time, and rP-8/4x concentration is shown in FIG. 3B. Thek_(obs) for the first and second step of the reaction are 1.69 and 0.05min⁻¹, respectively. The reaction is complete after 40 minutes and onlyrequires 20 nM ribozyme for maximal activity (with 1.33 nM substrate).These results show that the rP-8/4x ribozyme, in the absence of anucleotide cofactor, inherently contains the ability to bind a substratein trans and catalyze the TES reaction.

Even though there are 18 other uridines that could be sites of 5′cleavage and four other guanosines that could be sites of 3′ splicingfor the 36-mer substrate, only the expected TES product is generated.Apparently, the ribozyme recognition elements that define the individualcatalytic steps are sequence specific. That the ribozyme can catalyzethis reaction at all is surprising in that the relatively long bridgingregion could be expected to sterically hinder the binding of thesubstrate to the catalytic core of the ribozyme, or at leastsignificantly hinder the required conformational rearrangement betweenthe two catalytic steps. One or both of these might account for themajority (>50%) of the 36-mer substrate only undergoing the firstcatalytic step, in contrast to less than 10% only undergoing the firststep for the self-splicing reaction in vitro (15, 17). The lack ofproduct breakdown seen in the time dependence studies, however,indicates that the TES products themselves are not substrates forfurther reactions, although guanosine dependent 5′ cleavage orribozyme-mediated hydrolysis of the products could be a factor in vivo.

Previous studies utilizing the rP-8/4x ribozyme (Testa, S. M., Gryaznov,S. M. & Turner, D. H. (1998) Biochemistry 37, 9379-9385) show that the5′ exon mimic r(AUGACU) (SEQ ID NO:15) binds to the rP-8/4x ribozyme(K_(d)=5.2 nM at 37° C.) three orders of magnitude more tightly than the3′ exon mimic r(GUGCUCU) (SEQ ID NO:16) (K_(d)≈20 μM at 37° C.).Interestingly, maximum TES product formation occurs with as little as 20nM ribozyme (at 44° C.), indicating that for final product formation the5′ and 3′ exon intermediates produced during the 5′ cleavage step mightnot dissociate and then rebind the ribozyme before the exon ligationstep. To test for 5′ exon dissociation and rebinding between the twosteps, TES reactions were conducted with 166 nM rP-8/4x, 1.33 nMnon-radiolabelled 36-mer, and 1.33 nM radiolabeled 5′ exon, r(AUGACU)(SEQ ID NO:15). In this case, if the 5′ exon intermediate dissociatesfrom the ribozyme, the radiolabeled 5′ exon is just as likely to thenbind the ribozyme and form the 16-mer product as the non-radiolabeled 5′exon intermediate. As seen in FIG. 5, no radiolabeled TES products areobserved, indicating the 5′ exon intermediate does not dissociate fromthe ribozyme between the two steps (for those 5′ exon intermediates thatundergo the complete reaction).

Likewise, to test for 3′ exon intermediate dissociation and rebindingbetween the two reaction steps, TES reactions were conducted with 166 nMrP-8/4x, 1.33 nM radiolabeled 36-mer, and a 50 (66.5 nM) or 500 (665 nM)fold excess of a non-radiolabeled 3′ exon mimic competitor, r(GUGCUCU)(SEQ ID NO:16), which would form a 10-mer competition product. At equalmolar concentrations if the 3′ exon intermediate dissociates from theribozyme, the 7-mer competitor is 2.5 times more likely to bind theribozyme and be a substrate in the second reaction step than the 30-mer3′ exon intermediate (data not shown). The results (FIG. 5) show that a500-fold excess of cold competitor over substrate does not significantlyreduce the amount of 16-mer product formed (19.4%±2.3% versus 22.8%±3%,respectively). The small amount of 10-mer product that is observed at500-fold excess competitor over substrate (but not 50-fold excess) isnot actually competing with the TES reaction. In these cases, theribozymes that have bound radiolabeled 5′ exon regions, and for whichthe 3′ exon region has dissociated, are binding and reacting with asmall amount of the huge excess of 3′ exon competitor. Therefore, thevast majority of substrates that undergo the complete TES reaction donot have 3′ exon intermediate dissociation and rebinding occurringbetween the two steps of the reaction. Apparently, substrates thatundergo only the first reaction step do so because of nearlyirreversible 5′ or 3′ exon intermediate dissociation. It follows thatsince intermediates to the complete TES reaction do not dissociate fromthe ribozyme, the TES reaction is intramolecular with regard tosubstrate.

Example 3

Excision of a single nucleotide using the P. carinii. ribozyme. In orderto determine if a lower limit exists to the length of the excisedregion, we tested the TES reaction using the rP-8/4x ribozyme with twonew substrates. One substrate is a 12-mer, r(AUGACUGUGCUC) (SEQ ID NO:7), and was designed to contain the minimum length bridging sequencethat could utilize the 2 base pair RE2 interaction (to form the P9.0helix) and the 3′ guanosine thought to be required for self-splicing(FIG. 2Bi). The other substrate is a 10-mer, r(AUGACUGCUC) (SEQ ID NO:8), which can not utilize the RE2 interaction, and from which only onenucleotide would be excised (FIG. 2B ii). The results (FIG. 3A) showthat, under the optimal conditions of 10 mM MgCl₂ and 44° C., both the12-mer and 10-mer reactions lead to the formation of the expected 9-merproducts, as confirmed by enzymatic sequencing (data not shown). Theoptimized reactions produce 72%±3.9% product for the 12-mer substrateand 69.3%±4.4% product for the 10-mer substrate (for 6 independently runassays). Thus, the rP-8/4x ribozyme can excise as little as a singlenucleotide. The same approximate yield is obtained using the 12-mer and10-mer substrates which suggests that the role of forming the P9.0 helixis not large in this case. Therefore, the RE2 interaction, althoughperhaps beneficial, is not required for sequence specific TES reactions.In addition, the 12-mer and 10-mer substrates lead to more than twicethe product as compared with the 36-mer substrate, implicating thelonger bridging region (which includes the four 5′ bases of the intron)as being detrimental for this reaction. As the amount of substrate thatundergoes at least the first reaction step is similar for all of thedifferent substrates, 3′ exon intermediate dissociation for the 36-merlikely accounts for the difference in extent of final product formation.The dependence of the 10-mer substrate reaction on MgCl₂ concentration,time, and rP-8/4x concentration is shown in FIG. 3C. The k_(obs) for thefirst and second step of the reaction are 4.12 and 2.89 min⁻¹,respectively. In contrast to that for the 36-mer, the reaction with the10-mer substrate is more favorable at MgCl₂ concentrations greater than7 mM, and the second reaction step occurs approximately 50-fold faster.The origin of this effect is unknown, but could be due to the reducedsteric hindrance of the smaller bridge on the required conformationalrearrangement between the two reaction steps. This could reflect anincreased affinity or accessibility of the 3′ guanosine of the bridgefor the G-binding site of the ribozyme. Indeed, previous reports suggestthat the ability of the G-binding site to bind this endogenous guanosinedrives the second step of the reaction (Mahadevan, M., Tsilfidis, C.,Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang,M., Barcelo, J., O'Hoy, K., et al (1992) Science 255, 1253-1255; Harley,H. G., Rundle, S. A., MacMillan, J. C., Myring, J., Brook, J. D., Crow,S., Reardon, W., Fenton, I., Shaw, D. J., & Harper, P. S. (1993) Am. J.Hum. Genet. 52, 1164-1174).

Example 4

The sequence specificity of the P. carinii ribozyme can be altered. Forthe TES reaction to be useful, the recognition elements must be mutablein order to target predetermined sequences. Therefore, a truncated DMPKmimic model system was developed (FIG. 2Biii) for analyzing the abilityof a re-engineered ribozyme to excise the RNA triplet repeat regionwhose expansion is the causative agent of the effects of the geneticdisease, Myotonic Dystrophy. A ribozyme was designed to target theuridine immediately upstream of the repeats. Because there are fivesuccessive guanosines immediately following the repeats (which couldlead to a mixture of TES products), the first guanosine downstream fromthe five successive guanosines was targeted. The substrate bridgecontained 5 CUG repeats, as this is the smallest number of repeatsthought to form the hairpin structure similar to that seen with theexpanded transcripts. Myotonic Dystrophy patients actually have greaterthan 15 CUG repeats. Nevertheless, the ribozyme targets the flankingregions and would be the same regardless of the number of tripletrepeats. This ribozyme is referred to as the rP-8/4x-MD ribozyme andthis substrate the 38-mer DMPK mimic. The results at the optimum MgCl₂concentration (13 mM) and temperature (44° C.) are shown in FIG. 6A. A10 nucleotide product was obtained, as expected, in a yield of61.1%±4.6% (for 6 independently run assays). Besides unreacted 6-mergenerated from the first step of the reaction, no other products areproduced to any significant amount, indicating a reasonably specificreaction. The 10-mer product was extracted from the gel andenzymatically sequenced, along with a chemically synthesized version ofthe expected product. The sequence and banding patterns obtained (FIG.4) show that the expected TES product is being generated. Apparently,the ribozyme can be modified (at RE1, RE2, and RE3) to target non-nativesubstrates. The dependence of this reaction on MgCl₂ concentration,time, and rP-8/4x-MD concentration is shown in FIG. 6B. The k_(obs) forthe first and second step of the reaction are 0.41 and 0.44 min⁻¹,respectively (note that these values are within experimental error andthe rate of the second step is likely limited by the rate of the firststep). Interestingly, the excision of this 28-mer bridge, which couldinclude a triplet repeat hairpin structure, is substantially moreeffective than the 36-mer rP-8/4x system, which excises a 20-merunstructured bridge. Thus, targets with large bridge regions are notnecessarily poor reaction substrates.

To determine whether each ribozyme has specificity for its intendedtarget, TES reactions were run using the rP-8/4x-MD ribozyme with the36-mer P. carinii substrate at 13 mM MgCl₂, and the rP-8/4x ribozymewith the 38-mer DMPK mimic at 7 mM MgCl₂, each at 44° C. (FIG. 6A). Inaddition, the reaction was run using the rP-8/4x-MD ribozyme at 7 mMMgCl₂ and another reaction using the rP-8/4x ribozyme was run at 13 mMMgCl₂ (data not shown). In these reactions, not even 5′ cleavageproducts are observed, indicating that the ribozymes have somespecificity for their intended target substrates. Although the MgCl₂concentration required for maximum activity differs for all thereactions, they all occur at or below that required (15 mM) for maximumactivity for the P. carinii group I intron self-splicing reaction invitro. While it was known that the sequence of RE1 could be modified inreactions mimicking the first step of the self-splicing reaction (14,20, 21), it was previously not known that all three recognition elementsare modifiable. That these recognition elements completely specifybinding and reactivity indicates that they are the primary determinantsof specificity between the ribozyme and its substrate. In addition,bridging regions of different sequences and lengths (1, 3, 20, and 28nucleotides) have been excised, indicating that perhaps a 3′ G in thebridging region might be the only sequence requirement for the excisedsegment.

1. A method of removing a non-native nucleotide sequence from a targetnucleic acid sequence comprising contacting the target nucleic acidsequence with a modified trans-excision-splicing Group I ribozymecomprising at least two modifiable recognition elements, wherein atleast one of said recognition elements is complementary to thenon-native target sequence and at least one of said recognition elementsstabilizes binding of the ribozyme to a trans-excision splicing (TES)reaction intermediate product, and wherein the ribozyme catalyzes aspecific excision of the non-native target sequence in the absence of aguanosine cofactor and splices together the 5′ and 3′ ends of thesubstrate created by the excision.
 2. The method of claim 1 wherein thenon-native nucleotide sequence is a single nucleotide.
 3. The method ofclaim 1 wherein the non-native nucleotide sequence comprises a prematurestop codon.
 4. The method of claim 1 wherein the target sequencecomprises a frameshift mutation.
 5. The method of claim 1 wherein thenon-native nucleotide sequence comprises a triplet expansion repeatassociated with disease.
 6. The method of claim 5 wherein the disease isMuscular Dystrophy.
 7. The method of claim 6 wherein the ribozyme hasthe nucleotide sequence of SEQ ID NO:13.
 8. A method of removing aninternal expanded triplet repeat sequence from an RNA moleculecomprising contacting the RNA molecule with modifiedtrans-excision-splicing Group I ribozyme comprising at least twomodifiable recognition elements, wherein at least one of saidrecognition elements is complementary to non-native target RNA sequencewithin a substrate and at least one of said recognition elementsstabilizes binding of the ribozyme to a trans-excision splicing (TES)reaction intermediate product, and wherein the ribozyme catalyzes aspecific excision of the non-native target RNA sequence in the absenceof a guanosine cofactor and splices the substrate's 5′ end created bythe excision to the terminal G residue of the substrate's 3′ end createdby the excision.
 9. A method of removing a premature stop codon from amutant RNA sequence comprising contacting the mutant RNA sequence with amodified trans-excision-splicing Group I ribozyme comprising at leasttwo modifiable recognition elements, wherein at least one of saidrecognition elements is complementary to non-native target RNA sequencewithin a substrate and at least one of said recognition elementsstabilizes binding of the ribozyme to a trans-excision splicing (TES)reaction intermediate product, and wherein the ribozyme catalyzes aspecific excision of the non-native target RNA sequence in the absenceof a guanosine cofactor and splices the substrate's 5′ end created bythe excision to the terminal G residue of the substrate's 3′ end-createdby the excision.
 10. A method of removing a non-native nucleotidesequence comprising a triplet expansion repeat associated with MuscularDystrophy from a target nucleic acid sequence comprising contacting thetarget nucleic acid sequence with a modified trans-excision-splicingGroup I ribozyme having the nucleotide sequence of SEQ ID NO: 13 andcomprising at least two modifiable recognition elements, wherein atleast one of said recognition elements is complementary to thenon-native target sequence and at least one of said recognition elementsstabilizes binding of the ribozyme to a trans-excision splicing (TES)reaction intermediate product, and wherein the ribozyme catalyzes aspecific excision of the non-native target sequence and splices togetherthe 5′ and 3′ ends of the substrate created by the excision.